Hosts with established tumors fail to respond to tumor-associated antigens, and often appear to actively suppress responses to anti-tumor vaccines. This tumor-induced suppression constitutes a fundamental barrier to cancer immunotherapy. During the previous period of support, the project has identified the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) as occupying a pivotal role in coordinating this tumor-induced state of suppression and unresponsiveness. In mouse models, IDO expression directly suppresses CD8+ CTL responses, and potently enhances the suppressive activity of Foxp3+ Tregs. If IDO is inhibited using the IDO-inhibitor drug 1-methyl-tryptophan (1MT), hosts with established tumors become markedly more responsive to anti-tumor vaccines and T cell adoptive transfer. Further, when IDO is blocked, vaccine-induced inflammation becomes able to abrogate Treg-mediated suppression and re-program pre-existing Foxp3+ Tregs into pro-inflammatory T-helper/TH17 cells, resulting in significantly enhanced anti- tumor responses. The proposal will use models of defined TCR-transgenic T cells, therapeutic vaccination of large established tumors, and genetically-defined mouse strains, to test the following hypotheses: Aim 1 - Test the hypothesis that IDO-activated Tregs in established tumors and in vaccine-draining LNs mediate dominant suppression of vaccine-induced CTL and T-helper responses; but when IDO is blocked, vaccine-induced inflammation drives down-regulation of Treg-mediated suppression, re-programming of Tregs into polyfunctional T-helper cells, and IL-2-mediated enhancement of intratumoral CTL effector function. Aim 2 - Test the hypothesis that when IDO is blocked, CTLs become able to undergo full effector maturation in the tumor, mediate tumor-cell killing, and drive epitope-spreading to new CTLs against endogenous tumor antigens; but that activated CTL are still subject to PD-1-mediated mechanisms of clonal exhaustion within the tumor, and synergistically benefit from blockade of the PD-1 pathway. Aim 3 - Test the hypothesis that IDO inhibits CTL homing to the tumor by suppressing upregulation of chemokine receptor CXCR3 on activated CTLs, and by suppressing inflammation-induced VCAM-1 expression on tumor endothelial cells; but that inhibition of IDO allows therapeutic vaccination to create local inflammation within the tumor, driving active recruitment of anti-tumor effector cells. The long-term goal of these studies is to develop clinically-applicable combinations of an IDO-inhibitor drug plus therapeutic anti-tumor vaccines; and to use this combination to abrogate tumor-induced immune suppression and allow effective T cell responses against established tumors. The IDO-inhibitor drug 1-methyl- tryptophan (1MT) is currently entering Phase I clinical trials, so this strategy has high translational potential. PUBLIC HEALTH RELEVANCE: One of the fundamental barriers to successful immunologic therapy of cancer is the fact that tumors create a state of suppression and unresponsiveness to their own antigens. The natural immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) has been discovered to play a pivotal role in helping create this immune suppression. The long-term goal of the proposed studies is to develop treatment strategies based on the newly developed class of IDO-inhibitor drugs, using them in combination with therapeutic anti-tumor vaccines, so as to abrogate tumor-induced immune suppression and allow effective T cell responses against established tumors.